Oral drug administration is a noninvasive route of drug delivery for the treatment or prevention of diseased states in animals. The success of oral drug administration depends upon many factors, one of which is the degree of bioavailability of the drug. Bioavailability accounts for “the rate and extent to which the active ingredient or active moiety is absorbed from a drug product and becomes available at the site of action.” 21 C.F.R. § 320.1 (2000). In general, a host of biological factors affect bioavailability, including the pharmacokinetic behavior of the drug, drug formulation, site of administration, formulation and dosage form, and the physiological state of the patient.
In oral drug administration, one of the critical steps in determining bioavailability is the transportation and absorption of the drug across the various cell membranes comprising the intestinal mucosa (Lennernäs, H., “Human Perfusion Studies” in Oral Drug Absorption: Prediction and Assessment, Dressman, J. and Lennernäs, H., Eds., Marcel Dekker, 106, 99-117 (2000)). An important variable in calculating the extent of absorption is the effective permeability, Peff, because Peff is the rate-limiting step in the absorption process (Stein, W. D., Transport and Diffusion across Cell Membranes, Academic Press, Inc., Orlando, Fla., 1986).
Drug transport proceeds either by carrier-mediated transport, transcellular transport or passive diffusion mechanisms. Most orally-administered drugs are absorbed by passive diffusion mechanisms. For drugs that are absorbed by passive diffusion, there are a number of in vivo and in vitro intestinal absorption models that can be used to estimate the absorption of potential oral drug candidates during initial drug screening tests.
In order to accurately measure Peff, Lennerns et al. developed an in vivo human single-pass intestinal perfusion model to measure membrane transport. A large number of drugs belonging to several pharmacological classes were measured using this model, including cardiovascular system agents, nonsteroidal anti-inflammatory drugs (NSAIDs), central nervous system (CNS) agents, anti-infective agents, urinary tract system agents, and gastroenterological agents. These data serve as a guideline for classifying drug substances based on their aqueous solubility and intestinal permeability under the Biopharmaceutical Classification System (BCS) (fda.gov/cder/guidance/3618fnl.htm on the world wide web). The in vivo data generated by this method provide accurate Peff values, and are useful for comparative studies with in vitro data; however, the measurement of bioavailability by the human intestinal perfusion model or by other in vivo methods is impractical for HTS.
To this end, a number of in vitro absorption models have been developed to calculate and predict the parameters involved in drug absorption more quickly than in vivo models. These include the isolated intestinal cell model (Osiecka et al., Pharm. Res. 2, 284-293 (1985)), the everted intestinal ring model (Leppert and Fix, J. Pharm. Sci. 83, 976-981 (1994)), the everted intestinal sac model (Barthe et al., J. Drug Metab. Pharmocokinet. 23, 313-323 (1998)), the Ussing chamber model (Ussing and Zerahn, Acta. Phisiol. Scand. 23, 110-127 (1951)), the octanol-water partitioning model (Fujita et al., J. Am. Chem. Soc. 86, 5175-5180, (1964)), and the Caco-2 cell model (Artursson and Karlsson, Biochem. Biophy. Res. Comm. 175, 880-885 (1991)). Other models specifically utilizing chromatography methods include octadecyl-reversed phase chromatography (ODS) (Dorsey and Khaledi, J. Chromatography A 656, 485-499 (1993)), immobilized artificial membranes (IAM) (Yang et al., Advanced Drug Delivery Reviews 23, 229-256 (1996)), and micellar liquid chromatography (MLC) (Molero-Monfort et al., J. Chromatography A 870, 1-11 (2000)).
The accuracy of correlation of in vitro drug absorption values to those obtained by in vivo methods varies according to the model used. For example, one widely used in vitro model, the human intestinal Caco-2 cell line, emulates the intestinal cellular epithelium in humans very well (Artursson and Karlsson, Biochem. Biophy. Res. Comm. 175, 880-885 (1991)). Other in vitro models that may be used include the human intestinal epithelial cell lines, HT29-H and a co-culture of Caco-2 and HT29-MTX, wherein the outer surface of the cells contain a secreted mucin layer (Wikman et al., Pharmaceutical Research 10, 6, 843-852 (1993); Hilgendorf et al., J. Pharm. Sci. 89, 1, 63-75 (2000)). However, the experimental use of cell lines require costly and continuous maintenance programs to ensure cell viability. As a result, high performance liquid chromatography (HPLC) models, such as immobilized artificial membranes (IAM) and micellar liquid chromatography (MLC), which are experimentally easier to use than cell culture models, have been developed and shown to predict drug absorption with reliability and accuracy comparable to that of the Caco-2 cell model (Stewart et al., Pharm. Res. 15, 1401-1406 (1998)).
IAM creates mechanically stable chromatographic surfaces that mimic cell membranes (Pidgeon et al., Applications of Enzyme Biotechnology, 201-220 (1991)). The cell membrane structures are modeled after liposomes (Bangham et al., Methods Membr. Biol. 1, 1-68 (1974)) and are created by covalent attachment of the membrane-forming lipids, largely phospholipids, to a chromatographic surface.
U.S. Pat. No. 4,927,879 discloses a method for forming an IAM by the covalent attachment of amphiphilic cyclic dicarboxylic anhydrides to silica. The attached molecules form a tightly packed arrangement on the surface of the support to prevent additional nucleophilic reactions from occurring at these sites. U.S. Pat. No. 4,931,498 describes compositions and methods used to immobilize the membrane-forming lipids to silica supports.
Unlike IAM models, MLC models comprise a reversed stationary phase in combination with a surfactant solution mobile phase, wherein the surfactant concentration in the mobile phase exceeds its critical micelle concentration (CMC) (Escuder-Gilabert et al., J. Chromatography B 740, 59-70 (2000)). The surfactant, polyoxyethylene lauryl ether (Brij-35), adsorbs to the hydrophobic stationary phase and behaves as the polar membrane region of a cell. If supplemented with saline, the Brij-35 mobile phase further acts as an extracellular fluid. Like IAM models, MLC models predict the partitioning behavior of small molecule absorption into the lipid bilayers of the cell membrane.
IAM and MLC are currently being used to predict oral drug absorption and bioavailability. However, IAM and MLC methods are limited because they only model one part of the absorptive process, that is, the passive diffusion across the lipid bilayer. IAM and MLC do not model the initial absorption process across the epithelial mucosa, which is critical for drug absorption.
The epithelial mucosa, or mucus, is a hydrogel comprising inter alia, mucins, lipids, proteins, DNA, RNA and carbohydrates. The epithelial mucosa covers all epithelial surfaces, including respiratory, buccal, gastrointestinal, reproductive and urinary tract surfaces and coats the plasma membrane. Mucus functions to protect the cell surface from harmful extracellular molecules, and to regulate cellular interactions and molecular uptake that occur between the cell and its environment (Braybrooks et al., J. Pharm. Pharmac. 27, 508-515 (1975); Larhed et al., J. Pharm. Sci. 86, 660-665 (1997); Larhed et al., Pharm. Res. 15, 66-71 (1998)).
Mucins, the major components of mucus, are a family of high molecular weight glycosylated proteins that impart viscous and viscoelastic properties on mucus (Strous and Dekker, Critical Reviews in Biochemistry and Molecular Biology, 27, 57-92 (1992)). Differences in the type, level, and pattern of glycosylation in mucins is often altered in certain diseased states, including cancer (see, e.g., (Bhavanandan, Glycobiology 1, 493-503 (1991); Finn et al., U.S. Pat. No. 5,827,666). Mucins are primarily responsible for determining whether a substance, such as a drug, crosses the mucosal layer to enter the cells underlying the mucosal layer.
To date, in vitro models fail to provide a fast and convenient means to emulate the outer layer of the cell membrane and the cellular mucosa in order to investigate drug absorption. It would therefore be desirable to develop a chromatography model that measures the initial absorption process of a drug across a mimetic mucosal surface in order to obtain a more accurate representation of drug absorption across epithelial mucosa. Such a model would be particularly useful for modeling gastrointestinal drug absorption after oral administration. In addition, it would be desirable to develop a fast, cost efficient method to estimate drug absorption for HTS in both healthy and diseased states.